Plant chitinases belong to a family of pathogenesis-related (PR) proteins, which are over-expressed by plants in response to a pathogen attack (Giazinazzi, In Plant Microbe Interactions, Molecular and Genetic Perspectives, Vol. 1 (ed. Nester, E. W. & Kosuge T., 1987) 321–342; Boller, T., Id., Vol. 2, 385–413; Legrand, M., et al. Proc. Natl. Acad. Sci USA 84:6750–6754 (1987); Collinge, et al., Plant J. 3:31–40 (1993). Chitinases catalyze the hydrolysis of the β-1,4 linked N-acetylglucosamine polymers that form chitin chains, a major component of fungal cell walls. Chitinases have been divided based on their structure into at least four classes (classes i–iv). See, e.g., Hamel, et al., J. Mol. Evol. 44(6):614–24 (1997).
Even though chitinases have been shown to inhibit the hyphal tip growth of many fungi in vitro (Mauch, et al. Plant Physiol. 88:936–942 (1988)), a plant's natural defense mechanisms are often insufficient to prevent an invasion by the pathogen (Neuhaus, et al. Plant Mol. Biol. 16, 141–151 (1991)). The consequences of plant disease caused by fungal pathogens can be significant losses in crop quality and yields. Plants over-expressing chitinases under the control of a strong constitutive promoter have been engineered and have shown improved resistance against fungal pathogens under laboratory conditions (Broglie, et al. Science 254:1194–1197 (1991), Vierheilig, et al. Molecular Plant-microbe Interactions 6:261–264 (1993); Asao, et al. Plant Biotech. 14: 145–149 (1997); Tabei, Plant Cell Rep. 17: 159–164 (1998); Lorito et al., Proc. Natl. Acad. Sci. USA 95:7860–7865 (1998). Further, plants constitutively over-expressing a hybrid endochitinase exhibited improved tolerance to fungal diseases in field tests (Grison, et al. Nature Biotech. 14:643–646 (1996)).
The expression of chitinases in plants is therefore useful to enhance resistance in plants to fungi, including fungal pathogens. Chitinases expressed in plants are also reported to have anti-insect activity. See, e.g., Ding, et al., Transgenic Res. 7(2):77–84 (1998). Additionally, chitinases are useful in industrial processes aimed at the bioconversion of shellfish chitin waste (Cosio, et al. J. Food Sci. 47:901–905 (1982)).
In addition, nematode infection is a significant problem in the farming of many agriculturally significant crops. For example, soybean cyst nematode (Heterodera glycines, herein referred to as “SCN”) is a widespread pest that causes substantial damage to soybeans every year. Such damage is the result of the stunting of the soybean plant caused by the cyst nematode. The stunted plants have smaller root systems, show symptoms of mineral deficiencies in their leaves, and wilt easily. The soybean cyst nematode is believed to be responsible for yield losses in soybeans that are estimated to be in excess of $500 million per year. Other pathogenic nematodes of significance to agriculture include the potato cyst nematodes Globodera rostochiensis and Globodera pallida, which are key pests of the potato, while the beet cyst nematode Heterodera schachtii is a major problem for sugar beet growers in Europe and the United States.
Although expression of chitinases can be useful to reduce infection by fungal pathogens and other pests, constitutive overexpression of foreign proteins in crop plants has a potentially yield-reducing metabolic cost. Moreover, it is commonly found that particular chitinases only have antifungal activity against a narrow range of fungal pathogens. Further, it is known that certain nematodes, such as the soybean cyst nematode (“SCN”), can inhibit certain plant gene expression at the nematode feeding site. Thus, in implementing a transgenic approach to pathogen control, an important factor is to increase the expression of desirable genes in response to pathogen attack. Thus, chitinases with high activity and broad specificity are needed.